CN101367868B - Human Fc<gamma>R II linear ligand binding epitope - Google Patents

Abstract

The present invention relates to human FcγRII linear ligand binding epitope, the amino acid sequence of the epitope is Cys-Thr-Gly-Asn-Ile-Gly-Tyr-Thr-Leu-Phe-Ser-Ser-Lys-Pro-Val-Thr -Ile-Thr-Val, the present invention chemically synthesizes the linear ligand-binding epitope polypeptide of human FcγRII by using technologies such as bioinformatics, polypeptide synthesis, cellular immunology and molecular biology, and analyzes and identifies it. Inhibition of human IgG-soluble receptor binding and human IgG-cell surface receptor binding inhibition experiments showed that the linear epitope polypeptide showed good inhibitory effect on human FcγRII affinity human IgG, which is helpful for in-depth understanding of FcγR-IgG interaction The molecular basis of FcR is of great significance, and it provides new ideas for the molecular design of human FcR target drugs.

Description

Human FcγRII linear ligand-binding epitope

1. Technical field:

The invention relates to Fc receptors in the field of molecular immunology, in particular to human FcγRII (Fc GammaReceptor II) linear ligand-binding epitope.

2. Background technology:

Fc receptor (FcR) is a cell surface molecule with specific affinity for the Fc fragment of immunoglobulin (Ig). There are three types of human FcγR, namely FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Similar in structure, both contain two (FcγRII and FcγRIII) or three (FcγRI) Ig-like domains, belonging to the Ig supergene family, but there are significant differences in the transmembrane and intracellular regions, and Fc receptors are widely expressed in immune adjuvant Cells and effector cells have many important physiological functions, are the link between humoral immunity and cellular immunity, and play a key role in the immune regulation of the body. Antibody-mediated inflammatory responses can be regulated by activating and inhibitory FcRs, so FcRs are ideal drug targets for the treatment of allergies and autoimmune diseases.

According to the different functions of FcγR, increasing or decreasing the binding affinity of FcγR-IgG can achieve the corresponding purpose of FcγR immunotherapy. For the inhibitory receptor FcγRIIB, reducing the affinity of the receptor for IgG can lead to the loss of its inhibitory activity, thereby promoting the efficient presentation of tumor cells on antigen-presenting cells; inhibiting the expression of CD3 and CD4 on the cell surface with specific monoclonal antibodies Expression, and at the same time prevent it from combining with FcγRII and FcγRIII to prevent the body from producing an immune response, which can be used to prevent immune rejection caused by organ transplantation and inhibit autoimmune response. On the contrary, increasing the affinity of FcγRI can activate cellular immune function and enhance the antigen presentation of tumor cells. Soluble FcγR recombinant protein can block the binding of immune complexes to B cells in vitro, so it can compete with FcγR on the surface of immune cells to bind IgG in vivo, and inhibit the activation of cells by immune complexes; similarly, high doses of non-specific IgG can also It can compete with immune complexes in vivo to bind to immune cells expressing FcγR. Intravenous immunoglobulin therapy (IVIG) has been applied to thrombocytopenia (immune thrombocytopenic purpuria, ITP), systemic lupus erythematodes (systemic lupus erythematodes, SLE ) and multiple sclerosis (MS) and other diseases. The analysis of the crystal structure of IgG Fc-FcγRIII and the understanding of the signal transduction mechanism provide more information and ways to discover new specific and efficient drug targets. People use peptide library screening, combinatorial chemistry, and bioinformatics As well as synthetic polypeptides, a variety of inhibitory polypeptides of IgG Fc fragments were screened and identified. Among them, the TG19320 tripeptide tetramer that inhibits Fc-FcγR binding showed good inhibitory effect on glomerulonephritis in mice. Medgyesi et al. identified a functional polypeptide that induces cell signal transduction in the human IgG1 Fc fragment. The polypeptide derived from the Pro234-Ser298 of the CH2 region specifically binds to FcγRIIB on the cell surface, can induce cells to secrete cytokines and phosphorylate ERK, and can inhibit BCR. Mediated Ca ²⁺ response. However, the affinity of small molecular peptides to receptors is far lower than that of complete antibody molecules, and the improvement of peptide affinity has yet to be resolved.

3. Contents of the invention:

The technical problem to be solved by the present invention is to provide a human FcγRII linear ligand-binding epitope. In order to identify the inhibitory activity of the polypeptide of the present invention in vitro, the inhibition of the binding of the polypeptide to human IgG-soluble receptors and the binding of human IgG to human IgG were respectively carried out. -Inhibition of cell surface receptor binding, indicating that the linear epitope polypeptide exhibits good inhibitory efficacy on human FcγRII affinity human IgG.

Technical scheme of the present invention is:

The human FcγRII receptor linear ligand-binding epitope is characterized in that: the amino acid sequence of the epitope is Cys-Thr-Gly-Asn-Ile-Gly-Tyr-Thr-Leu-Phe-Ser-Ser-Lys-Pro- Val-Thr-Ile-Thr-Val.

The human FcγRII receptor linear ligand-binding epitope is a synthetic polypeptide.

The linear ligand-binding epitope is aminated or acetylated at the N-terminal of the polypeptide chain, or carboxylated or amidated at the C-terminal of the polypeptide chain.

The nucleotide sequence encoding the human FcγRII receptor linear ligand-binding epitope.

The present invention uses DNASIS software to compare and analyze the amino acid sequences of huFcγRIIA, boFcγRII, huFcγRIIB and moFcγRIIB, and design and synthesize 6 human FcγRII polypeptides in the EC2 domain with reference to the crystal structures of huFcγRIIA and huFcγRIIB. Dot-blot analysis shows that human FcγRII The 154-172 polypeptide CTGNIGYTLFSSKPVTITV is an effective polypeptide that specifically binds to human IgG. It is a linear ligand-binding epitope of human FcγRII and is located in the FG loop of the EC2 domain of the receptor. The results of polypeptide shortening show that the polypeptide CTGNIGYTLFSSK is the shortest and still has a similarity with human IgG. Peptides with strong binding activity.

In order to identify the inhibitory activity of the polypeptide of the present invention in vitro, the inhibition of the binding of the polypeptide of the present invention to human IgG-soluble receptors and the binding of human IgG-cell surface receptors were respectively carried out.

In the inhibition of the binding of the polypeptide of the present invention to human IgG-soluble receptors, the cDNA of the extracellular region of human FcγRII was subcloned into the prokaryotic expression vector pET-28a, and the extracellular region of human FcγRII was highly expressed in Escherichia coli for rapid dilution The renaturation method recovers the human FcγRII extracellular region recombinant protein with good binding activity from the denatured protein of the inclusion body, and uses the ELISA method to measure the inhibitory efficiency of the polypeptide of the present invention on human IgG-soluble receptor binding.

In order to inhibit the binding of peptides to human IgG-cell surface receptors, we expressed human FcγRII receptor molecules on the cell surface, subcloned the cDNA of the human FcγRII coding region into the expression vector pcDNA3, and transfected COS-7 cells. Through sex screening and continuous cloning, a human FcγRII transfection cell line stably expressing receptor molecules on the surface of COS-7 cells was obtained, and its IgG-RBC rosette formation rate was about 90%. The polypeptide of the present invention was detected by flow cytometry Inhibitory effect on human IgG-cell surface receptor binding.

Positive beneficial effect of the present invention:

1. This invention utilizes technologies such as bioinformatics, polypeptide synthesis, cellular immunology and molecular biology to chemically synthesize the linear ligand-binding epitope polypeptide of human FcγRII, and carry out analysis and identification. In the inhibition of receptor binding and human IgG-cell surface receptor binding inhibition experiments, the half-inhibitory dose IC ₅₀ can reach 112.3 μM, indicating that the linear epitope polypeptide has a good inhibitory effect on human FcγRII affinity human IgG.

2. The present invention is an exploration and research on the human FcR linear ligand binding epitope, which is of great significance for in-depth understanding of the molecular basis of FcγR-IgG interaction, and provides a new idea for the molecular design of human FcR target drugs.

3. The invention of the human FcγRII linear ligand-binding epitope polypeptide provides a new idea for finding drugs with high curative effect and small side effects to treat autoimmune diseases, and is of great significance to solve the autoimmune diseases that currently plague human health.

4. Description of drawings:

Figure 1: shows the alignment of the amino acid sequences of the FcγRII EC2 domain

Figure 2: It shows the double enzyme digestion identification and PCR identification of the prokaryotic expression plasmid pETshuRII

Figure 3: It shows the expression of pETshuRII/BL21(DE3) and its fusion expression detected by western blot

Figure 4: shows purification of expressed protein

Figure 5: It shows the activity of the refolding protein shuRII detected by ELISA

Figure 6: shows that R II6-C6 polypeptide inhibits the binding of human IgG to soluble human FcγRII

Figure 7: It shows the double enzyme digestion identification and PCR identification of the eukaryotic expression plasmid pc3huR II

Figure 8: It shows the identification results of monoclonal antibodies in human FcγRII transfected cells

Figure 9: It shows that the huFcγRII receptor molecule expressed on the surface of transfected cells can specifically bind to human IgG

Figure 10: shows that RII6-C6 polypeptide can inhibit the binding of human IgG to huFcγRII stably expressed on COS-7 cells

Five. Specific implementation methods:

Example 1: Design of huFcγRII polypeptide

Using DNASIS (Ver.2.5, Hitachi) software, the amino acid sequences of huFcγRIIA (NP_067674) and boFcγRII (NP_776964), huFcγRIIB (NP_003992) and moFcγRIIB (NP_034317) were compared and analyzed (Figure 1), and referred to huFcγRIIA (Maxwell et al. , 1999; Sondermann et al., 2001) and the crystal structure of huFcγRIIB (Sondermann et al., 1999), designed and synthesized 6 huFcγRII polypeptides, covering A-B, B-C, C-C', C'- E, E-F and F-G loops, except for huRII2 and huRII6 polypeptides that contain Cys at the N-terminal, Cys is added to the N-terminal of the other four polypeptides for carrier protein coupling (see Table 1).

Table 1 Characteristics of huFcγRII polypeptides and reactivity with human IgG

^a Cysteine residues are added in brackets at the N-terminal of the subsequent linking polypeptide sequence, ^b Dot-blot detection of the binding of each peptide to human IgG, "+" indicates that human IgG specifically binds to the peptide, "-" indicates that human IgG does not bind to the peptide, ^c the site predicted from the crystal structure of huFcyRII.

Example 2: Synthesis procedure and screening procedure of huFcγRII polypeptide

Peptides were synthesized by solid-phase peptide synthesis on Fmoc-Amino Acids attached to Wang Resin (Shanghai Jier) using a Symphony 12-channel peptide synthesizer (Protein Technologies Inc.). Synthesis was performed according to standard operating procedures. The specific process is as follows:

Design and synthesize peptide sequence→peptide program analysis→design synthesis program→calculate according to the substitution value of the resin and weigh Fmoc-AA-Wang-resin or Rink resin→DMF swelling resin→ ^* add 20% piperidine to remove Fmoc protection, stir for 6min→ ^* Add the next amino acid and HBTU for acylation reaction, N ₂ stirring reaction for 30min→ ^* Use Kaiser method or TNBS method to test the completion of the reaction→ ^* DMF wash 5 times, each 1min→Repeat the steps with ^* , in Connect the next amino acid to the resin until the synthesis of the polypeptide sequence is completed→select an appropriate reagent according to the amino acid composition of the peptide chain and use TFA to crack the connection between the peptide chain and the resin→precipitate the TFA polypeptide with cold ether→Sephadex G-25 desalting and purification→HPLC and LC-MS analysis to identify and purify the polypeptide→coupling of the polypeptide to the carrier→Dot-ELISA test for the binding of human IgG to the polypeptide→shortening of the ligand-binding epitope polypeptide→test the inhibition of the shortened polypeptide on the binding of human IgG-soluble receptor → Test the inhibition of binding of shortened polypeptides to IgG-cell surface receptors.

Both huFcγRII polypeptide and its shortened sequence are synthesized at 5-20 μmol/piece, and both contain Cys residues at their N-terminals, which can form disulfide bonds with SMCC bifunctional reagents and couple to carrier proteins. The synthetic polypeptide is identified by HPLC and LC-MS, and its structure is correct, and its purity can reach more than 80%.

Example 3: Coupling of huFcγRII polypeptide

，Pierce)通过将载体蛋白BSA上的-NH₂和多肽N端Cys的-SH相连，形成人工结合抗原多肽BSA-Pep。偶联步骤为：Application of heterobifunctional reagent Sulfo-SMCC (MW: 436.37, Spacer Arm Length: 11.6

, Pierce) by linking -NH ₂ on the carrier protein BSA with -SH on the N-terminal Cys of the polypeptide to form an artificial binding antigen polypeptide BSA-Pep. The coupling steps are:

，Pierce)，加入BSA溶液，充分混匀，室温反应1h或37℃30min，并不时混匀。(3)4℃对偶联缓冲液过夜透析，换液3次，去除多余偶联剂。该溶液即为SMCC活化BSA载体蛋白(SMCC-BSA)，用偶联缓冲液调整蛋白浓度为5mg/ml。(4)称取2mg多肽/条，以50μl二甲基甲酰胺(DMF)充分溶解，加入150μl含5mM EDTA的0.01MPB(pH7.2)，制备多肽储存液，浓度为10mg/ml。(5)偶联时，取10μl多肽储存液(100μg)，加入等体积含5mM EDTA的0.01M PB(pH7.2)；与20μlSMCC-BSA溶液(100μg)充分混合，室温反应4h，4℃过夜孵育。(6)以0.01M PB(pH7.2)调整偶联多肽浓度至1mg/ml，用于人IgG结合试验。(1) 4 mg of IgG-free bovine serum albumin (IgG-free BSA, Sigma-Aldrich) was weighed and dissolved in 0.5 ml of coupling buffer (0.1M PB pH7.2, 0.15M NaCl, 1 μM EDTA). (2) Dissolve 1 mg Sulfo-SMCC (MW: 436.37, Spacer arm length: 11.6

, Pierce), add BSA solution, mix thoroughly, react at room temperature for 1 h or 30 min at 37°C, and mix from time to time. (3) Dialyze the coupling buffer solution overnight at 4°C, and change the solution 3 times to remove excess coupling agent. The solution is SMCC-activated BSA carrier protein (SMCC-BSA), and the protein concentration is adjusted to 5 mg/ml with coupling buffer. (4) Weigh 2 mg of polypeptide/strip, fully dissolve it in 50 μl of dimethylformamide (DMF), add 150 μl of 0.01MPB (pH7.2) containing 5mM EDTA, and prepare a peptide stock solution with a concentration of 10mg/ml. (5) For coupling, take 10 μl of peptide stock solution (100 μg), add an equal volume of 0.01M PB (pH 7.2) containing 5 mM EDTA; mix well with 20 μl of SMCC-BSA solution (100 μg), react at room temperature for 4 hours, and overnight at 4°C Incubation. (6) Adjust the concentration of the conjugated polypeptide to 1 mg/ml with 0.01 M PB (pH 7.2) for human IgG binding test.

Example 4: Preliminary identification of the linear ligand-binding epitope of the huFcγRII receptor

1. Dot-blot

BSA-coupled polypeptide (1 μg/dot) was blotted on a nitrocellulose membrane (Millipore), and huFcγRII recombinant protein (shuRII) and carrier protein BSA were used as positive and negative controls; after natural drying, the blotted membrane was placed in a 0.2% Gelatin washing solution (0.05% Tween20, 0.01M PBS pH7.2, PBST) was blocked at 37°C for 1h; 10μg/ml HRP-labeled human IgG (HRP-IgG) solution diluted with 0.2% gelatin was added and incubated at 37°C for 1h to Fully washed with PBST; AEC (3-Amino-9-ethylcarbazole) color development kit (Zhongshan Jinqiao Co., Ltd.) was used to develop color according to the operation instructions. When judging the results, it was judged that the coated huFcγRII recombinant protein spot showed a brownish red color reaction, and the BSA control spot did not appear a color reaction, as shown in Table 1.

2. Precise mapping of huFcγRII linear ligand-binding epitope

In order to accurately locate the huFcγRII linear ligand-binding epitope, the amino acid residues of the huRII6 polypeptide were shortened one by one from the C-terminal, and a series of C-terminal truncated polypeptides were synthesized, and their binding ability to human IgG was detected by Dot-blot, as shown in Table 2. In the IgG-binding assay, the C-terminal truncated polypeptide RII6-C6 is the shortest polypeptide with strong binding activity to human IgG.

C-terminal truncated polypeptides of table 2 huRII6 polypeptides

Example 5: Inhibition of Human IgG-Soluble Receptor Binding by Linear Ligand-Binding Epitope Polypeptides

1. huFcγRII gene cloning

Human leukocytes were isolated from fresh anticoagulated human blood by referring to the method of separating bovine peripheral blood leukocytes by erythrocyte lysis method in the thesis of Zhang Gaiping. The source of this thesis is: Zhang G.1994.Bovine IgG Fc Receptors (PhD Thesis), University of Hertfordshire, Hatfield.

Total RNA was extracted with UNIQ-10 Column Trizol Total RNA Extraction Kit, and cDNA was synthesized from the isolated total RNA according to the instructions of the TAKARA 3’ FULL RACE kit. The reverse transcription system is 30 μl: Total RNA (about 1.5 μg) 4 μL, Oligo d(T)18 (50 pmol/μL) 3 μL, 5×M-MLV Buffer 6 μL, dNTPMixture (10mM each) 3 μL, RNase Inhibitor (40u/μL) 0.75 μL, Reverse TranscriptaseM-MLV (RNaseH) (200u/μL) 0.75 μL, RNase free Water 12.5 μL. After the above reaction solutions were mixed, the reverse transcription reaction was carried out in a PCR instrument according to the following procedure: 60 min at 42°C, 15 min at 72°C. Using the above cDNA as a template, using huR2A1 5`-CACAGTGCTGGGATGACTATGGAG-3` and huR2A2 5`-GACCACATGGCATAACGTTACTC-3` as primers, the full-length huFcγRII was amplified with LA Taq from Dalian Bao Biological Engineering Co., Ltd. (TAKARA), and the PCR reaction system was 25uL: cDNA 4μL, TAKARA LA Taq 0.25μL, 10×LA buffer 2.5μL, dNTP (2.5mM each) 2μL, huR2A1 (upstream primer) (20pmol/μL) 1μL, huR2A2 (downstream primer) (20pmol/μL) 1μL, Water14 .25 μL. The PCR conditions were 94°C for 3min for 1 cycle; 94°C for 30sec, 55°C for 30sec, and 72°C for 2min, a total of 35 cycles; 72°C for 10min. The PCR product was identified by 1% agarose gel electrophoresis, and the target fragment was recovered. And connect with pMD19-T vector, transform Escherichia coli, construct huFcγRII gene clone pTRII.

2.Expression of huFcγRII extracellular domain protein (shuRII) in Escherichia coli

2.1 Construction of the prokaryotic expression plasmid pETshuRII for the extracellular region of huFcγRII Using the pTRII plasmid as a template, primers PHR2-1-28a: 5′-TTT GAATTC GCTCCCCCAAAGGCTGTG-3′, PHR2-2-28a: 5′-TTT CTCGA GTGGTGA AGAGCTGCCCATG-3 ', synthesized by TAKARA company. The huFcγRII extracellular region gene was amplified with TAKARA Premix Taq, and EcoR I and XhoI restriction sites were introduced at both ends of the PCR product. The PCR reaction system is 50 uL: PremixTaq 25 μL, template DNA 2 μL, PHR2-1-28a (upstream primer) 2 μL, PHR2-2-28a (downstream primer) 2 μL, Water 19 μL. The PCR conditions were 94°C for 3min for 1 cycle; 94°C for 30sec, 55°C for 30sec, and 72°C for 1min, a total of 35 cycles; 72°C for 10min. The PCR product was identified by 1% agarose gel electrophoresis, and the target fragment was recovered and digested by EcoR I and Xho I. The obtained target fragment was inserted into the corresponding restriction site of the expression vector pET28a, and the recombinant pET28a was transformed into the expression strain BL21(DE3) , Picked a single clone to extract the plasmid for enzyme digestion identification, and named it pETshuRII.

2.2 Expression of ShuRII protein:

Transform the positive recombinant plasmid into BL21(DE3) expression host bacteria, pick a single colony, and inoculate it into 5ml LB (containing 100μg/mL kanamycin) culture medium with shaking at 37°C overnight, and use 10% inoculum volume the next day Transfer to 5ml of LB medium (containing 100μg/mL kanamycin), cultivate to OD600nm about 0.6, add IPTG (final concentration: 1mmol/L) for induction, then culture at 37°C for 3-7h, centrifuge ( 4000r/min×20min) to collect the bacteria, resuspend the bacteria in PBS containing 400μg/ml lysozyme, lyse the bacteria in a water bath at 30°C for 30min, and ultrasonically treat the lysed bacteria solution for 99×3s, intermittently for 8s (ice bath), 4°C Centrifuge at 12000r/min for 20min, separate the supernatant and precipitate of the cell lysate, and identify the expression result by SDS-PAGE.

2.3 Western blot detection:

After 12% SDS-PAGE of pETshuRII whole bacteria induced by IPTG and uninduced whole bacteria, the electrophoresis-separated protein was charged and transferred to PVDF membrane, blocked with 0.2% gelatin overnight, and the membrane was washed 6 times in PBST, 3 min each time, Add 1:500 times diluted horseradish peroxidase-labeled human IgG and incubate at 37°C for 1 h, wash the membrane with PBST 6 times, each time for 3 min, and detect the target protein by AEC color development.

2.4 Inclusion body purification:

Induce the expression of 250ml bacterial culture, and collect the expressed bacteria by centrifugation. After sonication in 20ml lysis buffer (10mM Tris PH8.0, 150mM NaCl, 10mM EDTA, 10% glycerol, 2mM DTT, 0.5mM PMSF, 400μg/ml lysozyme), the inclusion body pellet was collected by centrifugation. Wash the inclusion bodies 5 times with wash buffer (1% Triton-100, 50mM Tris PH8.0, 100mM NaCl, 10mM EDTA, 2mM DTT), 25ml each time; then use resuspension buffer (50mM Tris PH8.0, 100mM NaCl, 10mM EDTA, 2mM DTT) washed once, and finally centrifuged to precipitate the purified inclusion body.

2.5 Refolding of ShuRII protein:

Dilution refolding method: wash and purify inclusion bodies with 2ml guanidine buffer (6mol/L guanidine hydrochloride, 50mmol/L Tris-C1pH8.0, 10mmol/L EDTA, 10mmol/L DTT, 100mM NaCl, 10% glycerol) in Stir at 4°C for about 10 hours to dissolve the denaturation, remove the insoluble matter by centrifugation, then dilute the shuRII denaturation solution to 5ml with guanidine buffer, and place it at room temperature for 0.5-1h; add the denaturation solution to 250ml of refolding buffer dropwise in four batches 100mM Tris pH8.0, 400mM L-arginine hydrochloride, 2mM EDTA, 5mM reduced glutathione, 0.5mM oxidized glutathione, 0.1mM PMSF, 0.1mM sodium azide), the final concentration is 0.1mg/mL, each interval of 12h, so that the refolding time reaches 60h. Centrifuge 250ml of the refolding solution at 8000r/min at 4°C for 25min, take the supernatant, and use a 5kDa MWCO concentrator tube to concentrate the supernatant refolding solution to the desired concentration.

2.6 ELISA identification activity:

The microtiter plate was coated with 10 μg/mL shuRII protein before and after refolding, and blocked with 0.2% gelatin. Add different concentrations of horseradish peroxidase-labeled human IgG (HRP-huIgG) (0.01-25μg/ml) solutions to the coated plate, and detect human IgG and shuRII on the microplate before and after renaturation Protein binding, compare the activity of protein before and after refolding. BSA, which does not bind to human IgG, was used as a negative control protein.

2.7 Inhibition of Human IgG-Soluble Receptor Binding by Linear Ligand-Binding Epitope Peptides

Coat the microtiter plate with 10 μg/ml shuRII recombinant protein and block with 0.2% gelatin. Mix different concentrations of polypeptides (0.1-70μM) with an equal volume of 10μg/ml HRP-IgG solution, and react at 4°C for 2 hours; add the polypeptide-IgG mixture to the shuRII recombinant protein-coated plate, and detect human IgG and ELISA plate The above-mentioned shuRII recombinant protein binding was used to determine the inhibitory efficiency of the polypeptide on shuRII protein binding to human IgG. A polypeptide that does not bind human IgG and an irrelevant protein BSA served as negative controls.

3. Results

3.1 Construction of pETshuRII expression vector

Using the recombinant plasmid pTRII as a template, the huFcγRII extracellular region gene was amplified with primers PHR2-1-28a and PHR2-2-28a, and the huFcγRII extracellular region gene was directional inserted into the expression vector pET-28a by EcoR I and XhoI double digestion . After PCR identification and enzyme digestion identification, the results showed that the huFcγRII extracellular region gene had been directionally inserted into the expression vector, and the recombinant expression plasmid pETshuRII was obtained, as shown in Figure 2.

Figure 2 shows the double enzyme digestion identification and PCR identification of the prokaryotic expression plasmid pETshuRII, Lanes in the figure: M1.DL2000DNA marker; 1. The PCR product of the extracellular region of huFcγRII (537bp); 2. pETshuRII was digested by EcoRI and XhoI The product (5369bp, 537bp); M2.λDNA/HindIII DNA marker (23130, 9416, 6557, 4361, 2322, 2027, 564bp).

3.2 Induced expression, solubility analysis and Western blot identification of ShuRII protein

The expression products after IPTG induction for 3h, 5h, and 7h were subjected to polyacrylamide gel electrophoresis (SDS-PAGE), and there was an obvious characteristic protein expression band at about 24.8kD, and the protein expression level tended to increase after IPTG induction for 5h. more stable. The bacteria were disrupted by ultrasonication and centrifuged to obtain the supernatant and precipitate. No expression product was seen in the supernatant, indicating that the recombinantly expressed protein in vitro mainly existed in the form of inclusion bodies (Fig. 3A). The expressed protein showed a Western blot with a size of 24.8kD on the PVDF membrane, but there was no bacterial lysate before induction with the recombinant plasmid, indicating that the expressed protein could specifically bind to human IgG ( FIG. 3B ).

Figure 3 shows the expression of pETshuRII/BL21(DE3) and its fusion expression detected by immunoblotting. In the figure: (A) SDS-PAGE analysis of protein expression. Lanes: M. Protein standard control; 1. Bacteria before recombinant plasmid induction Lysate; 2. Bacterial lysate after recombinant plasmid induction for 3 hours; 3. Bacterial lysate after recombinant plasmid induction for 5 hours; 4. Bacterial lysate after recombinant plasmid induction for 7 hours; 5. Bacterial lysate supernatant after recombinant plasmid induction; 6. Precipitation of bacterial lysate after recombinant plasmid induction;

(B) Western blot analysis of protein specificity.

3.3 Purification of ShuRII protein

According to literature reports, a denaturing solution with an alkaline pH value is beneficial to renaturation. We used pH 8.0 buffer solution to purify shuRII inclusion bodies by ultrasonic washing six times. SDS-PAGE analysis showed that the purity of inclusion bodies after washing can reach more than 90%. , as shown in Figure 4, shows that the washing effect is better, which lays the foundation for the future renaturation. Figure 4 shows the purification of expressed proteins. In the picture: Lanes: M. Protein standard control;

1. pETshuR II/BL21(DE3) cells lyse insoluble inclusion bodies;

2. The purified product after the first washing of the inclusion body;

3. The purified product after the third washing of the inclusion body;

4. The purified product after the fifth washing of the inclusion body;

5. The purified product after the sixth washing of the inclusion body.

3.4 ELISA detection of refolding protein activity

The purified protein is further diluted and refolded to obtain a proper spatial structure. In order to detect the binding activity of the refolding protein shuRII and the ligand, we coated the microtiter plate with 10 μg/ml of the protein (inclusion body) after refolding and before refolding, and used different concentrations of HRP-huIgG (0.01-25 μg/ ml) for detection.

As shown in Figure 5, it shows that the ELISA detects the activity of the refolding protein shuRII. It can be seen from the figure that the difference between the OD values of the two ELISAs becomes significantly larger with the increase of the ligand concentration, indicating that the dilute refolding has obtained Active protein (results are the average of triplicate experiments).

3.5 Inhibition of Human IgG-Soluble Receptor Binding by Linear Ligand-Binding Epitope Peptides

The RII6-C6 polypeptide was used to interact with HRP-IgG, and its inhibitory effect on the binding of soluble huFcγRII to human IgG was determined. In the competition ELISA, the RII6-C6 polypeptide can inhibit the binding of human IgG to soluble huFcγRII, and the inhibitory ability is enhanced with the increase of the concentration of the RII6-C6 polypeptide, but it cannot be completely inhibited. Binding of HRP-IgG to soluble receptors had no significant effect. Figure 6 shows the inhibition of RII6-C6 polypeptide on the binding of human IgG to soluble human FcγRII, and the results are the average of three repeated experiments.

Example 6: Inhibition of Binding of Linear Ligand-Binding Epitope Polypeptides to IgG-Cell Surface Receptors

1. Construction and identification of eukaryotic recombinant expression vectors:

Using the pTRII plasmid as a template and primers PCR2-1: 5′-ATA GAATTC GCTGGGATGACTATGGAG-3′, PCR2-2: 5′-GGG CTCGAG CATAACGTTACTCTTTAG-3′, the full-length huFcγRII coding region (ORF) was amplified with TAKARA Premix Taq cDNA (including signal peptide sequence), PCR reaction system is 50uL: Premix Taq 25μL, template DNA 2μL, PCR2-12μL, PCR2-22μL, Water 19μL. The PCR conditions were 94°C for 3min for 1 cycle; 94°C for 30sec, 55°C for 30sec, and 72°C for 1min, a total of 35 cycles; 72°C for 10min. The PCR product was identified by 1% agarose gel electrophoresis, and the target fragment was recovered and digested by EcoR I and Not I. The obtained target fragment was inserted into the corresponding restriction site of the expression vector pcDNA3, and the recombinant pcDNA3 was transformed into E.col i JM109 sensory State cells were cultured on nutrient agar plates containing 50 μg/mL ampicillin. Pick a single clone to extract the plasmid for enzyme digestion and identification, and named it pc3huRII.

2. Cell Transfection

2.1 Plasmid preparation Qiagen plasmid extraction kit was used to extract and purify eukaryotic expression plasmid pc3huRII. The pc3huRII plasmid was digested with BglII, identified by 1% agarose gel electrophoresis, and the recovered target fragment was dissolved in sterile water, and the DNA content was determined with a nucleic acid protein analyzer (Beckman Company). The linearized expression plasmid pc3huRII was used for stable expression of huFcγRII.

2.2 Cell transfection and screening COS-7 cells were transfected with linearized pc3huRII plasmid using liposome Lipofectamine ^TM 2000 transfection reagent. Digest the transfected cells 48 hours after transfection, resuspend the cells in 20 mL of DMEM/10 complete medium containing 400 μg/mL G418, then transfer to a 96-well culture plate, 200 μL per well, and culture in a 50 mL/L CO2 incubator at 37 °C. Change the medium every 3 to 4 days. 7 to 10 days after transfection, when the cell clone grows to 1/4 to 1/2 well, digest the cell clone, transfer the cell to another 96-well culture plate for expansion culture, and wait for the cell monolayer to grow into a monolayer, and immunofluorescence The expression of huFcγRII was detected, and the cells in the original 96-well plate were retained for further culture.

2.3 Cloning of transfected cells The positive transfected cell clones were cloned three times consecutively by the limiting dilution method, and a cell line stably expressing huFcγRII was established. Transfer the positive cell clones detected by immunofluorescence to a 24-well culture plate for expanded culture, dilute the cells to 5-10 cells/mL with DMEM/10 selection medium containing 200 μg/mL G418, inoculate a 96-well cell culture plate, 200 μL per well, Cultivate in a 50mL/L CO2 incubator at 37°C for 10-15 days, and detect the expression of huFcγRII by immunofluorescence. The huFcγRII-positive cell clones were cloned twice and three times, so that the positive rate of immunofluorescence in the clone wells reached more than 85%, and the cloned cells were frozen in liquid nitrogen.

3. Immunofluorescence detection

COS-7-huFcγRII cells transfected with pcDNA3/huFcγRII were planted in a 96-well plate at a density of 1×10 ⁵ , and COS-7 cells not transfected with pcDNA3/huFcγRII were used as a control, and the cells were buffered with PBS after 24 hours. washed with blocking solution (0.002 g/mL gelatin) for 30 min, incubated with mouse anti-human CD32 monoclonal antibody (20 μg/mL) for 1 h at 4°C, and rinsed gently with PBS for 6 times; The anti-mouse IgG was incubated with the secondary antibody in the dark for 1 h, and after washing with PBS buffer, the expression of huFcγRII on the cells was observed with a laser scanning confocal microscope.

4. Rosette test

Refer to the rosette formation method in Zhang Gaiping's paper, the source of which is: Zhang G.1994. Bovine IgG Fc Receptors (PhD Thesis), University of Hertfordshire, Hatfield., pp. 54-58.

The binding of huFcγRII to its ligand human IgG on the surface of COS-7 transfected cells was detected by rosette assay. Sheep erythrocytes (SRBC) were treated with tannic acid to make 50mL/L erythrocyte suspension with PBS. Take 1mL of 50mL/L tannic acid-treated SRBC and mix with the same amount of 200μg/mL human IgG, and mix and incubate at room temperature for 45min , washed 3 times with PBS, and finally made into 10mL/L human IgG-SRBC. Culture huFcγRII transfected cells in a 96-well plate, and when the cells grow into a monolayer, rinse the cells with pre-warmed serum-free DMEM/0 medium (37°C), add DMEM/0 medium, 200 μL per well, and incubate at 37°C 2h, to elute the bovine IgG bound to the cell surface; slowly add 10mL/L human IgG-SRBC suspension to the cell monolayer, 50μL per well; incubate at 37°C for 10min, let stand at room temperature for 35min; gently rinse with PBS 6 times ; Fix the cells with methanol (containing 3 mL/L H ₂ O ₂ ) at room temperature for 10 min, rinse with PBS 3 times; add 100 μL of PBS to each well, and observe the rosettes formed on the cell surface under a microscope. Add 10 μg/mL HRP-labeled goat anti-human IgG antibody to the cell monolayer, 50 μL per well, and incubate at 37°C for 30 min; wash thoroughly with PBST, stain with AEC staining kit; observe the color development of SRBC in the rosette with a microscope.

5. Inhibition of IgG-cell surface receptor binding by linear ligand-binding epitope polypeptides

Prepare the COS-7 cell suspension expressing huFcγRII, and adjust the cell concentration to 1×10 ⁷ /mL. Dilute the RII6-C6 polypeptide with DMEM/0 medium (containing 0.1% sodium azide), mix it with an equal volume of human IgG-FITC suspension respectively, and react at 4°C for 1 h. RII6-C6 polypeptide with different concentrations (0.1 -700μM) bound to human IgG; add the RII6-C6-IgG-FITC mixture to the cell suspension stably expressing huFcγRII, react at 4°C for 1 hour, wash 3 times and use flow cytometry to detect the inhibitory effect, and set up a random combination The polypeptide and unrelated protein BSA were used as negative controls.

6. Results

6.1 Construction of eukaryotic expression plasmid pc3huRII

Using the positive plasmid cloned on the T vector as a template, the full-length 971bp cDNA (including signal peptide) sequence of the huFcγRII coding region (ORF) was amplified with primers PCR2-1 and PCR2-2. The full-length cDNA of huFcγRII was subcloned into the downstream of the cytomegalovirus promoter (Pcmv) of pcDNA3, and the eukaryotic expression plasmid pc3huRII was successfully constructed after PCR identification and EcoR I and Xho I double enzyme digestion identification. The identification results are shown in Figure 7, Fig. 7 shows the double enzyme digestion identification and PCR identification of the eukaryotic expression plasmid pc3huR II.

In Figure 7: M1: λ-Hind III digest Marker (23130, 9416, 6557, 4361, 2322, 2027, 564bp); 1: pc3huRII digested by EcoR I and Xho I;

2: PCR product (971bp) amplified with primers PCR2-1 and PCR2-2;

M2: DL2000 Marker (2000, 1000, 750, 500, 250, 100bp).

6.2 Establishment of transfected cell lines stably expressing huFcγRII

COS-7 cells were transfected with the linearized expression plasmid pc3huRII. After G418 selective culture, immunofluorescence detection and serial cloning, a transfected cell line stably expressing huFcγRII was established. About 90% of the transfected cells showed fluorescence. See Fig. 8A; while no fluorescence was seen in the untransfected COS-7 control cells, see Fig. 8B, indicating that the huFcγRII receptor molecule was effectively expressed on the surface of the transfected cells.

Figure 8 shows the results of immunofluorescence detection of stably transfected pcDNA3/huFcγRII cells. In the figure A: COS-7 cells transfected with pc3huRII (200×); B: non-transfected COS-7 cells (200×).

6.3 Binding of huFcγRII to its ligand on the surface of COS-7 transfected cells

Transfected cells expressing huFcγRII receptor molecules were tested with human IgG-SRBC for the rosette test, and obvious rosettes were formed around the transfected cells, while untransfected COS-7 control cells did not bind to human IgG-SRBC and had no rosettes Figure 9 shows the formation of huFcγRII receptor molecules expressed on the surface of transfected cells can specifically bind to human IgG. In the figure A: COS-7 cells transfected with pc3huRII (200×); B: non-transfected COS-7 cells (200×).

6.4 Inhibition of IgG-cell Surface Receptor Binding by Linear Ligand-Binding Epitope Peptides

Different concentrations of RII6-C6 polypeptides interacted with human IgG-FITC, and the inhibitory effect of RII6-C6 polypeptides on the binding of human IgG to huFcγRII was determined on stably expressing huFcγRII transfected cells. The results of flow cytometry showed that the RII6-C6 polypeptide could inhibit the binding of human IgG to huFcγRII, and the inhibitory ability was enhanced with the increase of the concentration of the RII6-C6 polypeptide, with an IC ₅₀ of 112.3 μM; There is no inhibitory effect (the result is the average value of three repeated experiments), see Figure 10.

sequence listing

<110> Henan Academy of Agricultural Sciences

<120> Human FcγRII linear ligand-binding epitope

<130> Method for Polypeptide Synthesis

<160>1

<170>PatentIn version3.4

<210>1

<211>19

<212>PRT

<213> Artificial sequence

<400>1

Claims (5)

1. people's Fc γ RII acceptor linear ligands combination epitopes, it is characterized in that：The amino acid sequence of the epitope is Cys-Thr-Gly-Asn-Ile-Gly-Tyr-Thr-Leu-Phe-Ser-Ser-Lys-Pro- Val-Thr-Ile-Thr-Val.

2. people's Fc γ RII acceptor linear ligands combination epitopes according to claim 1, it is characterized in that：People's Fc γ RII acceptor linear ligands combinations epitope is synthesis polypeptide.

3. people's Fc γ RII acceptor linear ligands combination epitopes according to claim 1, it is characterized in that：Described linear ligands combination epitope is in the amination of polypeptide chain N-terminal or acetylation.

4. people's Fc γ RII acceptor linear ligands combination epitopes according to claim 1, it is characterized in that：Described linear ligands combination epitope is in polypeptide chain C-terminal carboxylated or amidatioon.

5. encode the nucleotides of people's Fc γ RII acceptor linear ligands combination epitopes described in claim 1.

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